The Rad52 SSAP superfamily and new insight into homologous recombination

Rad52 is a highly conserved eukaryotic protein that can mediate the annealing of complementary DNA strands to initiate homologous recombination for the repair of double-strand breaks1. Suspicions that at least some prokaryotic single-strand annealing proteins (SSAPs) are related to Rad52 have been discussed for more than two decades. Two recent cryo-EM structures2,3 now put the issue beyond doubt.

proposition is now conclusively established and a new class of protein fold has been identified (Fig. 1a).
The RAD52 SSAP superfamily protein fold involves five conserved elements: (i) an antiparallel three stranded β-sheet, which forms the inner surface of the helical filament; (ii) an α-helix (α3 in Fig. 1) that is packed across the three β-sheet strands. Together α3 and the β-sheet are the most conserved part of this protein fold (blue in Fig. 1) and set the inherent curvature of the filaments formed by multimerization; (iii) a second α-helix (α2, green) packs with α3; followed by (iv) a β-hairpin and (v) α-helix 1. Utilizing AlphaFold 14,15 for SSAP modelling revealed that not only are other RecT/Redβ SSAPs based on the same design but also members of the Erf group (Fig. 1b), as previously anticipated 12 . All SSAPs contain the first three elements but variability in the other two elements (Fig. 1b, grey) is evident. Notably the most diverse Rad52 members, S.c.Rad59 16 and the prokaryotic SakRad52 17 , appear to either lack the β-hairpin or present a greatly extended version of it. To further validate the Rad52 superfamily, we screened the AlphaFold library of one million structures using Foldseek 18 with the five-element structure from HsRAD52. Of the 25 top hits, 18 were Rad52 variations (and another 5 were unknown proteins). Therefore, we conclude that the Rad52 SSAP fold is not common or previously identified and probably unique to SSAPs. This list of 25 plus a variety of AlphaFold SSAP structures for viewing by PyMOL can be found at this link https:// sharing.biotec.tu-dresden.de/index.php/s/8cZI7i8EdZhENoN The new bacteriophage filament structures not only secure the Rad52 SSAP superfamily hypothesis, but also deliver pioneering insight into SSAP annealing mechanisms, because no highresolution structure of RAD52, or any other SSAP, bound to annealed DNA strands has been previously achieved. In concordance with a previous deduction 11 , the annealed DNA strands lie on the outside of the helical filament and the two strands do not cross each other with respect to the underlying protein helix. Therefore, despite Watson-Crick base pairing, the annealed strands must be more underwound than B-form dsDNA. This explains how SSAPs can be tightly bound to the annealed intermediate but have no, or little, affinity for B-form dsDNA.
Both bacteriophage structures show one DNA strand bound in a deep groove through electrostatic and hydrogen bonds to the phosphodiester backbone so the bases are presented outward. This groove is the same as the known RAD52 ssDNA binding groove 19 and includes the only identifiable amino acid sequence signature in the Rad52 SSAP superfamily 11 . However, concordant with the substantial divergence of amino acid sequence, the binding of the ssDNA in the groove differs in detail. For example, LiRecT presents a repetitively kinked five nucleotide/monomer regularity whereas Redβ and HsRAD52 appear to be 4 nucleotides/monomer. Notably in both cases the bound ssDNA is stretched about 1.5 fold, which is the same as ssDNA stretching by RecA/Rad51. The second ssDNA strand is bound to the first by Watson-Crick base pairs with little evidence for extensive binding into a second groove or trans interactions with another filament. So it is unlikely that the second strand is stretched before annealing. Consequently, the homology search by SSAPs is likely to be similar to the stretched versus unstretched search mechanism utilized by RecA/Rad51, where an initial match can be found and then expanded as the second strand is zipped into position through Watson-Crick pairing 20 . The evidence for a cisbased zipping mechanism concords with observations from atomic force microscopy and optical tweezer single-molecule studies with Redβ, which also revealed a substantial increase in complex stability upon the annealing of~10 bases (now revealed to be dimerization of Redβ) and a transition to a remarkably stable complex, termed a DNA clamp, resistant to 200 pN of pulling force 11,21 . The basis for a DNA clamp is evident in both of the new bacteriophage filaments, however apparently involving different secondary structural elements that move to secure the DNA after annealing. Once again, the principle appears to be the same however the details are different. RecT. e AlphaFold projected full-length Redβ filament displaying electrostatic surfaces presenting the positively charged ssDNA binding groove (red) between negatively charged ridges (blue). The C-terminal three α-helical bundle 27 , which is not part of the annealing domain or the published cryo-EM structure 2 but is required for HR 23 , is the perpendicular projection away from the helical axis of the filament.

Outlook
The perception that all SSAPs are ancestrally anchored in the Rad52 superfamily promotes functional implications. Notably, helical filaments have not been reported for RAD52 rather only rings that may be heptamers 8 or undecamers 9,10 . Despite this evidence that RAD52 multimerization is flexible, rather than a cis-zipping mechanism, ring-to-ring trans-annealing models have been favoured 19,22 . In light of the new SSAP structures, a reappraisal of the RAD52-annealing mechanism may be rewarding.
Recent progress with the simpler HR Redβ mechanism 23,24 could also illuminate Rad52 action. Both Rad52 and Redβ annealing domains, which like all members of the Rad52 SSAP superfamily occupy~180 amino acids at the N-terminus, are insufficient for HR and protein-protein interactions with their C-terminal regions are required 23,25 . One of these interactions involves the major cellular single-strand binding protein, termed replication protein A (RPA) in eukaryotes and single-strand binding (SSB) in prokaryotes 26 . For eukaryotic Rad52, the RPA interaction with a specific Rad52 C-terminal region was defined some time ago 25 . Interaction between the C-terminus of λ phage Redβ and E.coli SSB was recently identified by inspired deduction 27 . Concomitantly the first functional evidence for SSB contribution to phage SSAP-mediated HR was reported 28 . This emergent commonality involving eukaryotic Rad52/RPA and prokaryotic phage SSAPs/SSB is another indicator that Rad52 and phage SSAP HR mechanisms are related. Consequentially, now that the Rad52 SSAP superfamily is secured, a new light is cast on Rad52 action and the vast diversity of prokaryotic SSAPs can be confidently evaluated for structural and mechanistic variations around a central theme.